Response to Reviewers
We would like to thank the reviewers for the their time and constructive
comments. Please see find below our responses to their comments.
Reviewer: Hanna Rennert
Major points
1. It is not clear from the results how successful the BMT was. Figure 1
demonstrates results of PCR analysis for the sry gene, but does not provide
any quantitative information regarding the success rate. The authors should
be more specific regarding the number of animals and the number of cells that
successfully engrafted.
We used an established protocol that gives approx 80% donor chimerism as
measured by GFP in peripheral blood (Li et al JASN 2006), Body weight was
also monitored and in the event of weight loss or absent/weak sry detection of
engraftment, mice were excluded. Ultimately the detection of BM-derived cells
in the kidney is a measure of successful engraftment (eg fig 4c). Numerous
BM derived cells were found and analyzed which was the aim of the
experiment. Reference to our previous experience with bone marrow
transplantation and monitoring body weight is now included in methods and
results.
2. In Figure 3 the authors show that the percentage of Y chromosome-positive
cells is declining in the kidney. Was that decline also observed in the animals'
peripheral blood and how do the authors explain this decline?
This is probably due to the resolution of the inflammatory infiltrate. The main
point that we are aiming to make in this figure is that BM-derived cells were
readily detectable at the each of the time points examined.
3. This sentence in the Results section “These frequencies and total numbers
of Y chromosome-positive cells observed are consistent with previous reports
of BM-derived cells on the tubule [21]” should be moved to the Discussion.
The authors should also clearly state the mean percentages of Ychromosome positive cells in the peripheral blood and kidneys as well as the
number of animals assessed.
We do not have peripheral blood data for these experiments, but when we
have monitored engraftment using GFP in the past approximately 80% of
leukocytes in peripheral blood are donor derived. This previous work is now
cited [24]. Numbers of mice assessed are in the methods and have been
added to the fig legend – “Sections assessed were from 2 mice a 2 weeks, 3
mice at 4 weeks and 2 mice at 12 weeks.”
4. The authors note in the Results that BM-derived cells were only found as
scattered examples in the renal tubule. The low number of these cells in the
kidney may reflect unsuccessful transplant which would be a limitation of this
work, but this is not addressed in the Discussion and needs to be added.
Cells were scattered in the kidney, but were not rare indicating robust
engraftment (Figure 4c). It was only in the tubule that BM-derived cells were
uncommon, but this is as previously reported by other investigators.
5. The authors also indicate that the longer time point (twelve weeks)
assessed is sufficient for the post-ischaemic recovery and the development of
cystic pathology, however, this may not be sufficient time for complete
engraftment, particularly since no information is provided regarding the
success of the BM transplant. BM cells contribution to epithelial cells following
HSCT is well documented, including in humans. However this may be
depended on level of engraftment. This should be discuss in more details.
Repopulation by transplanted bone marrow in the irradiated mouse occurs
rapidly. Six weeks was allowed for engraftment prior to surgical induction of
injury and then a further 12 weeks recovery in our longest time point. Previous
studies investigating BM-derived epithelial cells in the kidney have examined
mice 4 to 8 weeks after BM transplantation (eg Lin et al 2005 J Clin Invest
115:1756-1764., Duffield et al 2005 J Clin Invest 2005, 115(7):1743-1755.)
Minor points
6. Introduction- ADPKD is caused by inherited mutations in one of two genes:
PKD1 and PKD2, followed by secondary somatic mutations in the renal
epithelial cells leading to cysts development. This is not indicated in the text
and should be added.
A sentence explaining this has been included in the introduction.
“In ADPKD, cysts have been reported to originate from the clonal expansion
of epithelial cells with an inherited mutation plus an acquired somatic mutation
of one of two ADPKD-related genes (PKD1 or PKD2).”
7. Results, page 8- “Detection rates were not 100% because the Y
chromosome is not necessarily be included in a cross-section of every
nucleus examined”. “ the Y chromosome is not necessarily included…”?
Text corrected.
“ Detection rates were not 100%, even in male control kidney, because the Y
chromosome is not always present in the remaining portion of sectioned
nuclei.”
8. Results, page 9- “From 120 examples of Y chromosome positive cells
examined from kidney 2, 4 or 12 wks after injury, only a 10 (2 at 2wk, 5 at 4
and 3 and 12wk) were sufficiently entwined with the tubule that an epithelial
phenotype might be considered”. Check sentence. “…only 10 examples…”?
Text corrected.
“From 120 examples of Y chromosome positive cells examined, only 10 cells
were sufficiently entwined with the tubule that an epithelial phenotype might
be considered (2 cells at 2 weeks, 5 cells at 4 weeks and 3 cells at 12
weeks).”
9. Discussion, page 12- “Many of the initial studies in this area appear to have
overestimated the contribution of BM-derived cells to renal epithelia due to
unreliable tracing and phenotype determination techniques.. Provide
references.
Duffield et al 2005 JCI [19] is now cited.
.
10. Manuscript should be check carefully for typos and grammar. There are
numerous typos throughout the text: Introduction-2nd paragraph change
“donour” to “donor”. Figure legend 3: change “inury” to “injury”; Figure legend
6: change “modelling” to “modeling”, “epithithelial” to “epithelial”.
Typos have been corrected and spelling Americanized
.
Reviewer: Anna Paola Paola Mitterhofer
Major Compulsory Revisions
1. In the paper the aim of the study is not clearly expressed and the
assumptions described in the background don’t bind together. Moreover the
key points are not correct. Authors describe the study but not the objectives of
the study. Authors assess that “In light of reports cells derived from
hematopoietic stem cells in the bone marrow (BM) can integrate into the
kidney and form epithelial cells” referring to papers published in 2001. They
don’t consider that it has been widely demonstrated in subsequent studies
that the role of bone marrow-derived cells to epithelial integrity restoration
after an ischemic insult is very poor and not significant. (Reference 15.,16.,
Kidney tubular epithelium is restored without replacement with bone marrowderived cells during repair after ischemic injury Duffield JS, Bonventre JV.
Kidney Int. 2005 Nov;68(5):1956-61). Authors assess that “the reoccurrence
of PKD has not been reported in a genetically normal renal graft” but
nevertheless they decide to use PKD (Polycystic Kidney Disease) mutant
bone marrow-derived cells to evaluate PKD cell capability of expressing the
disease phenotype in ischemic genetically normal kidney. This to explain a
phenomenon which notoriously doesn’t happen.
While there is a consensus that BM-derived cells are not major contributors to
epithelial repair in the kidney, low level contributions have been reported in
several recent studies. More recent reports of BM-derived epithelial cells
include (Lin et al 2005 JCI; Yang et al J Am Soc Nephrol 22: 2028–2036,
2011, Syres et al 2009 Blood 114: 2542-2552, Li et al 2012 Am J Phys Renal
302:F9-F19). These studies are now cited in the manuscript. To our
knowledge, there has been no specific examination of bone marrow-derived
cells carrying a PKD causing in the transplanted human kidney. While
macroscopic manifestations of engrafted PKD cells can presumably be
excluded, the existence and phenotype of rare tubular cells could be
informative with regard to the process of cystogenesis. We reasoned that it
would be worthwhile to investigate this question in a controlled mouse model
where tissue preparation and cell detection techniques could be optimized.
2. methods applied seem to be well conduct referring to a previous study of
the same authors on post- ischemic acute tubular necrosis. Authors show
many nice figures, well described.
3. part of methods are described in background and should be shifted. (from
“We used a mouse model to test whether mutant…. to “to induce transplanted
BM cells to home to the kidney and give rise to renal epithelial cells”.
We feel that this needs to stay in the introduction as it gives an overview of
our experimental approach. Specific details are in the methods section.
4. the data reported are well described but the research doesn’t constitute a
useful contribution to the field
The examination of putative BM-derived epithelial cells in the kidney has been
complicated by a lack of unambiguous markers. We have use bone marrow
with a mutation causing epithelial overproliferation, a phenotype that has the
potential to identify a genuine epithelial contribution. We also address the
issue of the stage of repair by examining three time points. Additionally we
note a qualitative difference in the Y-chromosome signal from epithelial and
interstitial cells that is informative.
5. data consist in the careful description of pathology results of the experiment
conducted obtained step by step, with three-dimensional reconstruction and
description
6. most conclusions are foregone Authors discuss data already known in
literature, related to the role of BM-derived cells in ischemia restoration, but
they refer to these reports only in the discussion and conclusion. This means
that study design is completely unfounded
References 9, 10, 11, 12, 13, 16, 18, 19, 20 refer to the BM-derived cells and
ischemia (or acute renal failure) and are included in introduction. Reference
20 has been added to the introduction and is a review reinforcing the opinions
(that BM does not give rise to epithelium) expressed in reference 19.
7. limitations of the work are not clearly stated
We discuss the rarity of BM-derived cells in the renal tubule, the difficulty of
unambiguously determining the identity of BM-derived cells in the renal
tubule, and the possibility of BM-derived cells fusing with the renal tubule.
8. title and abstract accurately convey what has been found. Authors avoid
the ischemic field already explored with BM-derived cells
This field is discussed and referenced throughout (References 9, 10, 11, 12,
13, 16, 18, 19, 20). Additional relevant references have been added including
some more recent reports (references 14, 15, 16)
9. the writing is acceptable